Complex comprising a psma-targeting compound linked to a lead or thorium radionuclide

ABSTRACT

The present invention relates to complexes comprising a prostate-specific membrane antigen (PSMA) targeting compound linked to a radionuclide, such as 212Pb or 227Th, through a TCMC or DOTA chelating moiety. These compounds, and pharmaceutical compositions comprising them, can be used for medical applications. These applications include the treatment of prostate cancer, and the complexes allow for dual targeting of cancers.

FIELD OF THE INVENTION

The present invention relates to complexes comprising a PSMA targetingcompound linked to a radionuclide such as ²¹²Pb or ²²⁷Th. Thesecompounds, and pharmaceutical compositions comprising them, can be usedfor medical applications. These applications include the treatment ofprostate cancer, and the complexes allow for dual targeting of cancers.Peptide and peptidomimetic PSMA targeting urea derivatives conjugated tochelators for complexing ²¹²Pb and ²²⁷Th for radiotherapeutic use. Thesecan be used in single and dual targeting.

BACKGROUND OF THE INVENTION

Prostate cancer is among the most frequent causes of cancer relatedmortality in men. There is a great demand for new and effectivetreatment, especially in hormone refractory late stage disease. Skeletalmetastases are a frequent problem in late stage disease and thereforethe alpha-particle emitter ²²³Ra (Xofigo) was introduced as a bonespecific therapy for late stage prostate cancer patients with skeletalmetastases.

Although, as a bone-seeker, ²²³Ra shows significant clinical benefit forpatients its activity is limited to the bone metastases and is nottargeting soft tissue metastases.

Several carrier molecules for radioligand targeting of prostate specificmembrane antigen (PSMA) exists. Lutetium-177 labeled PSMA-617(¹⁷⁷Lu-PSMA-617) is the compound in most advanced clinical developmentstage for use in radionuclide therapy.

This molecule works in a suitable manner and give relevant tumor tonormal tissue ratios for longer lived (i.e. a half-life of a few days)radionuclides, including ¹⁷⁷Lu and ²²⁵Ac, but at early times points(typically a few hours after injection) shows high uptake in kidneys.With shorter lived radionuclides like ²¹²Pb (half-life of 10.6 hours),the initial kidney uptake represents a potential toxicity problem.

It is therefore advantageous to use a PSMA-ligand with less kidneyuptake, but this should not compromise the tumor uptake. The PSMA ligandmolecules are made up of (1) a PSMA-binding region, (2) a linker regionand (3) a chelator, whereby the linker region connects the (1) and (3).The linker region also is used to adjust molecular size and polarity etcto affect the in vivo distribution properties. The PSMA-binding region(motif) used in PSMA-617 is a structure that can be found in severalmolecules of this class, developed by several different inventors andresearchers, including PSMA-11 and PSMA I&T as well as ¹³¹I and ²¹¹Atlabelled PSMA binding ligands.

New compounds that contain a PSMA region are warranted because atcurrent all ligands in testing have challenges, including a relativelylow radiobiological effectiveness (RBE) and suboptimal biodistribution.

There is also a need for an improved alpha emitter that can target boththe bone metastases and the soft tissue metastases.

The present invention relates to compounds that addresses thesechallenges.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to the complex of the presentinvention, wherein the compound X is linked to a radionuclide, such as²¹²Pb or ²²⁷Th, by a chelating moiety Z.

In one embodiment of the present invention is the radionuclide ²¹²Pb.

In another embodiment of the present invention is the radionuclide²²⁷Th.

The chelating moiety Z may be selected from the group consisting ofacyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrinsor cyclic or noncyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate,DOTA, a DOTA derivative, pamidronate conjugated to DOTA, TCMC, a TCMCderivative, pamidronate conjugated to TCMC, antibody-conjugated-DOTA,antibody-conjugated-TCMC, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO,DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A.

In one embodiment of the present invention is the linker DOTA or a DOTAderivative.

In another embodiment of the present invention is the linker TCMC or aTCMC derivative.

For ²²⁷Th are octadentate hydroxypyridinone-containing ligands, such as3,2-HOPO, particularly suitable.

An aspect of the present invention relates to the radiopharmaceuticalcomposition according to the present invention for use as a medicament.

An aspect of the present invention relates to the radiopharmaceuticalcomposition according to the present invention for use in the treatmentof soft tissue and or skeletal disease.

In one embodiment of the present invention is the skeletal diseaseselected from the group consisting of skeletal metastases from cancersto the breast, prostate, kidneys, lung, bone, or multiple myeloma, ornon-cancerous diseases causing undesired calcification includingankylosing spondylitis.

FIGURES

Figure text

FIG. 1 shows biodistribution of ²¹²Pb 2 hours after injecting²¹²Pb-labeled p-SCN-Bn-TCMC-PSMA ligand 1 and PSMA-617 respectively, inthe presence of ²²⁴Ra.

FIG. 2 shows survival of mice with C4-2 PSMA positive xenograft aftertreatment with saline, 52 MBq ¹⁷⁷Lu-OSMA-617 and 0.32 MBq²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1.

DETAILED DESCRIPTION OF THE INVENTION

Some abbreviations used

Peptide mimetic—also termed peptidomimetic, is a small protein-likechain designed to mimic a peptide. They typically arise either frommodification of an existing peptide, or by designing similar systemsthat mimic peptides, such as peptoids and β-peptides. Irrespective ofthe approach, the altered chemical structure is designed toadvantageously adjust the molecular properties such as, stability orbiological activity. This can have a role in the development ofdrug-like compounds from existing peptides. These modifications involvechanges to the peptide that will not occur naturally (such as alteredbackbones and the incorporation of nonnatural amino acids). Based ontheir similarity with the precursor peptide, peptidomimetics can begrouped into four classes (A-D) where A features the most and D theleast similarities. Classes A and B involve peptide-like scaffolds,while classes C and D include small molecules.

PSMA—Prostate-specific membrane antigen. Synonyms PSMA, ProstateSpecific Cancer Antigen, PSM, FGCP, FOLH, GCP2, mGCP, GCPII, NAALAD1,NAALAdase, FOLH1, Glutamate carboxypeptidase 2, Glutamatecarboxypeptidase II, Membrane glutamate carboxypeptidase,N-acetylated-alpha-linked acidic dipeptidase I,Pteroylpoly-gamma-glutamate carboxypeptidase, Folylpoly-gamma-glutamatecarboxypeptidase, Folate hydrolase 1, Prostate-specific membraneantigen, Cell growth-inhibiting protein 27

DOTMP—1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonicacid)

EDTMP—ethylenediamine tetra(methylene phosphonic acid)

EDTA—ethylenediaminetetraacetic acid

p-SCN-Bn-DOTA—2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid

DOTA-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and alsoused for benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(e.g. conjugated to monoclonal antibody)

p-SCN-Bn-TCMC—2-(4-isothiocyanotobenzyl)-1, 4, 7, 10-tetraaza-1, 4, 7,10-tetra-(2-carbamonyl methyl)-cyclododecane

TCMC—1, 4, 7, 10-tetraaza-1, 4, 7, 10-tetra-(2-carbamonylmethyl)-cyclododecane and also used for benzyl-1, 4, 7, 10-tetraaza-1,4, 7, 10-tetra-(2-carbamonyl methyl)-cyclododecane (e.g. conjugated tomonoclonal antibody)

mAb—monoclonal antibody.

HOPO—Me-3,2-HOPO, octadentate hydroxypyridinone for complexing 227Th,4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoicacid and derivatives.

Ligand 1—p-SCN-Bn-TCMC-PSMA ligand 1, p-SCN-Bn-TCMC-PSMA etc.

Ligand 2—p-SCN-Bn-DOTA-PSMA ligand 2, p-SCN-Bn-DOTA-PSMA etc

The same abbreviations are in the following used for acids, salts orpartly or fully dissociated versions of the chelators.

The present invention relates to single and dual targeting propertysolutions based on a small molecular urea derivative as a prostatecancer cellular targeting agent for carrying ²¹²Pb.

This may be used with purified ²¹²Pb or in a dual targeting solutionwhereby ²²⁴Ra will act as a skeletal treatment and²¹²Pb-urea-deriviative will act as a systemic therapy againts cellsexpressing PSMA antigen which is associated mainly with advancesmetastatic prostate cancer, and to some extent also other types ofcancer.

It is known in the field that urea based compounds conjugated to achelator group can facilitate the targeting of radionuclides to PSMAexpressing cells. Radionuclides that have been evaluated forradiotherapeutic purposes with PSMA targeting includes ¹⁷⁷Lu, ²¹¹At,²¹³Bi, and ²²⁵Ac.

The invention is in the field of radiolabeled therapy agents. Accordingto the invention, radiolabelled derivatives of urea basedprostate-specific membrane antigen (PSMA) inhibitors are disclosed.

Thus, the present invention relates to a complex comprising a compound Xlinked to a radionuclide, such as ²¹²Pb or ²²⁷Th, wherein the compound Xis a peptide or peptidomimetic urea derivative suitable for targeting ofPSMA expressing cells and tissues.

Linker

An aspect of the present invention relates to the complex of the presentinvention, wherein the compound X is linked to a radionuclide, such as¹⁷⁷Lu, ²¹³Bi, ²²⁵Ac, ²¹²Pb or ²²⁷Th, by a chelating moiety Z.

In one embodiment of the present invention is the radionuclide ²¹²Pb.

In another embodiment of the present invention is the radionuclide²²⁷Th.

In another embodiment of the present invention is the radionuclide¹⁷⁷Lu.

In a further embodiment of the present invention is the radionuclide²¹³Bi or ²¹²Bi.

In yet another embodiment of the present invention is the radionuclide²²⁵Ac.

It is to be understood that the complexing agent, or linker, orchelating moiety Z, according to the invention may also coverderivatives of the above-mentioned compounds (such as derivatives ofEDTMP, DOTA, for example p-SCN-Bn-DOTA and TCMC, for examplep-SCN-Bn-TCMC). It is of course to be understood that such derivativesmust maintain the capability to complex ²¹²Pb with a higher stabilityconstant than to ²²⁴Ra.

The chelating moiety Z may be selected from the group consisting ofacyclic chelators, cyclic chelators, cryptands, crown ethers, porphyrinsor cyclic or noncyclic polyphosphonates, DOTMP, EDTMP and bisphosphonatederivatives, DOTA, a DOTA derivative such as p-SCN-Bn-DOTA, pamidronateconjugated to DOTA, TCMC, a TCMC derivative such as p-SCN-Bz-TCMC,pamidronate conjugated to TCMC, antibody-conjugated-DOTA,antibody-conjugated-TCMC, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO,DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A.

In one embodiment of the present invention is the linker DOTA or a DOTAderivative, such as p-SCN-Bn-DOTA.

In another embodiment of the present invention is the linker TCMC or aTCMC derivative, such as p-SCN-Bn-TCMC.

The complexing agent may be linked via the carbon backbone allowing all“binding arms” of the chelator molecule interact with the metal.Alternatively, one of the arms may be used as a linker.

Suitable chelators include DOTA derivatives such asp-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (p-SCN-Bz-DOTA) and DOTA-NHS-ester.

Thus, for p-SCN-Bn-DOTA or p-SCN-Bn-TCMC can the complexing agent belinked via the carbon backbone (C-backbone) to the rest of the compound.

In one embodiment is the linker an octadentatehydroxypyridinone-containing ligands, such as 3,2-HOPO. Such ligandswill typically comprise at least one chelating group of the followingsubstituted pyridine structure (I):

-   -   17-R, I

Wherein R, is an optional N-substituent group and may thus be absent ormay be selected from hydrocarbon, OH, O-hydrocarbon, SH andS-hydrocarbon groups, where any or each hydrocarbon moiety isindependently selected from short hydrocarbyl groups, such as C1 to C8hydrocarbon, including C1 to C8 alkyl, alkenyl or alkynyl groups, or maybe an OH or O-hydrocarbon. R, may also comprise a linker moiety, asindicated below and/or may comprise a coupling moiety as also indicatedbelow. In Formula I, groups R2 to R6 may each independently be selectedfrom H, OH, ═O, short hydrocarbon (as described herein), a linker moiety(as described herein) and/or a coupling moiety (as described herein).Generally, at least one of groups R, to R6 will be OH. Generally, atleast one of groups R2 to R6 will be =0.

Generally, at least one of groups R, to R6 will be a linker moiety.Preferably, exactly one of groups R2 to R6 will be =0. Preferablyexactly one of groups R, to R6 will be OH. Preferably exactly one ofgroups R, to R6 will be a linker moiety (as described herein). Theremaining groups R, to R6 may be any of those moieties indicated herein,but are preferably H. Where a linker moiety or any additional linker,template or chelating groups attached to a linker moiety do not comprisea coupling moiety then one of groups R, to R6 is preferably a couplingmoiety (as described herein).

In a preferred embodiment, one of groups R, to R6 will be OH and one ofR2 to will be =0 and the OH and =0 groups will be on neighbouring atomsof the ring. Thus, in a preferred embodiment, OH and =0 may be on atoms1,2; 2,3; 3,2; 3,4; or 4,3 respectively (numbering from the nitrogen aswould be expected).

Octadentate ligands having at least one chelating moiety wherein OH and=0 groups are present at positions 3 and 2 respectively are highlypreferred. The octadentate ligands may have 2, 3 or 4 such chelatinggroups, where 2 or 4 such groups are highly preferred.

In a special embodiment an urea derivative based PSMA targeting complexis labeled with ²¹²Pb or ²²⁷Th in a mixture of bone-seeking ²²⁴Ra or²²³Ra, respectively, for dual targeting by (1) PSMA cellular targetingand (2) targeting of skeletal metastases related bone synthesis byradium cations.

Complexing agents for ²²⁷Th including those described in WO2011098611,US20170319721, Ramdahl et al. (Bioorganic & Medicinal Chemistry LettersVolume 26, Issue 17, 1 Sep. 2016, Pages 4318-4321), and Hagemann et al.(Mol Cancer Ther. 2016 October; 15(10):2422-2431. Epub 2016 Aug. 17)conjugated to an urea derivative for PSMA targeting. The complexingagents mentioned herein are hereby incorporated by reference.

Thus, one embodiment of the present invention relates to a PSMAtargeting urea derivative comprising a TCMC group, such asp-SCN-Bn-TCMC, for chelating ²¹²Pb.

Another embodiment of the present invention relates to a PSMA targetingurea derivative comprising HOPO for chelating ²²⁷Th.

A further embodiment of the present invention relates to a PSMAtargeting urea derivative comprising DOTA, such as p-SCN-Bn-TCMC,labeled with either ²¹²Pb or ²²⁷Th.

In yet an embodiment, the complexing agent does not complex ²²⁴Ra orsubstantially complex ²²⁴Ra in the pharmaceutical composition.

In yet a further embodiment, the complexing agent complexes with ahigher stability constant to ²¹²Pb than to ²²⁴Ra.

In an embodiment, the stability constant for ²¹²Pb is at least twice theaffinity for ²²⁴Ra, such as at least four times higher, such as at least8 times higher or such as at least 10 times higher.

In an embodiment said complexing agent is selected from the groupconsisting of, ligand-conjugated-DOTA, such asligand-conjugated-p-SCN-Bn-DOTA, or ligand-conjugated-TCMC, such asligand-conjugated-p-SCN-Bn-TCMC.

The ligang may be an antibody or polypeptide.

In a further embodiment, the amount of ²²⁴Ra and ²¹²Pb is in radioactiveequilibrium.

In yet a further embodiment, the activity ratio (in MBq) between ²¹²Pbto ²²⁴Ra is between 0.5 and 2, such as 0.8-1.5, or such as 0.8-1.3, orpreferably such as 0.9-1.15.

In the present context, the term “radioactive equilibrium” relates tothe ratio in MBq between two radionuclides being the same orsubstantially the same over time. The term “activity ratio” e.g. between²¹²Pb and ²²⁴Ra relates to the ratio of MBq of ²¹²Pb to ²²⁴Ra. In FIG. 5is a table (table 2) showing the development of this activity ratio overtime. It can be seen that after two days a radioactive equilibrium of1.1 has been established for the activity ratio between ²¹²Pb to 224 Ra(7.3 divided by 6.8). Thus, in FIG. 5, it can also be seen that theradioactive equilibrium between ²¹²Pb and ²²⁴Ra is reached after about 2days.

In the present context, the terms “complexing agent”, “scavenger”,“linker”, “chelating moiety Z”, and “chelating agent” are usedinterchangeably. The terms relate to agents capable of forming complexeswith ²¹²Pb, preferably by chelation and with a significant strength asmeasured in test systems while radium is not significantly affected bythe presence of the complex as measured in the test systems.

Test systems include in vivo biodistribution and in vitro cationexchanger or size retention and centrifuge concentration cartridge forchelate-antibody binding of radionuclide. Alternatively, thin layerchromatograpy may be used as a test system.

In the present context “scavenging” (or complexing) is defined as atleast 50% bound according to thin layer chromatography (TLC), centrifugeconcentration separation or bio-distribution profiles.

This means, as an example, at least 50% less blood uptake of ²¹²Pb witha small molecular chelator. With an antibody-conjugated chelator, whereblood uptake is not a reliable indicator, at least 50% bound accordingto TLC analyses.

In one embodiment of the present invention is at least 60% bound.

In another embodiment of the present invention is at least 70% bound.

In another embodiment of the present invention is at least 80% bound.

In another embodiment of the present invention is at least 85% bound.

In another embodiment of the present invention is at least 90% bound.

The compound or compounds may also be capable of complexing moreradionuclides than ²¹²Pb.

In one embodiment of the present invention, the compound and/or complexis at a concentration of 1 ng/mL to 1 g/mL.

In another embodiment of the present invention, the compound and/orcomplex is at a concentration of 100 ng to 10 mg/mL

The complex can comprise one, two, three, four, five or more compounds.

In one embodiment is the solution in a volume of 100 μL to 1000 mL, suchas 500 μL to 100 mL, 1 mL to 10 mL.

In one embodiment of the present invention is the radioactivity of thesolution 1 kBq to 1 GBq, such as 10 kBq to 100 MBq, such as 100 kBq to10 MBq.

In another embodiment of the present invention is the radioactivity ofthe solution 100 kBq to 100 MBq.

In another embodiment of the present invention, the complexing agent isconjugated to a compound selected from the group consisting of peptidesand peptide mimetic urea derivatives with affinity for PSMA.

In another embodiment of the present invention, the complexing agent isthe chelator TCMC, such as p-SCN-Bn-TCMC, or DOTA, such asp-SCN-Bn-DOTA, conjugated to a compound selected from the groupconsisting of a monoclonal antibody, a polyclonal antibody, an antibodyfragment, a synthetic protein, a peptide, a hormone or hormonederivative or a vitamin or a vitamin derivative.

For dosing purposes, a pure ²¹²Pb solution may be used. Alternatively,²²⁴Ra in mixture with ²¹²Pb may be used, the latter for dual targetingpurposes, i.e., ²²⁴Ra targets bone metastases and ²¹²Pb targets systemiccancer by its urea derivative carrier. If ²²⁴Ra comprising solutions areadministered they may have been stored for some time, e.g., 1 day ormore preferably at least two days, such as 1-2 days or 1-3 days, toreach equilibrium between ²²⁴Ra and ²¹²Pb/²¹²Bi. This will ensure ²¹²Pbto ²²⁴Ra activity ratios between 0.83 and 1.14. This can, e.g., beaccomplished by the manufacturer by simply retaining the product for aday or so before shipment.

Alternatively, ²¹²Pb may be added to ²²⁴Ra solutions to obtain aspecific radionuclide ratio. E.g. if the soft tissue tumor burden ismuch higher than the skeletal tumor burden, a pure ²¹²Pb preparation ora solution with a high ²¹²Pb to ²²⁴Ra ratio may be used.

The non-overlapping side effect profile of cationic radium and alphaemitters conjugated to PSMA binding urea-derivatives makes a mixture of²²⁴Ra cation and ²¹²Pb-PSMA targeting agent for dual targetingparticularly attractive. This is because more modest dosing of eachcompound can be used to produce antitumor activity, since, at least forthe skeletal component, the two different compounds will target thelesions independently.

It is important to keep radium as mainly un-complexed, or weaklycomplexed cation as this ensures a maximum uptake in bone and bonemetastases and also ensures favourable excretion of eliminated productmainly through the intestines.

By adding complexing agent to a solution of radium the radioactivedaughter can be made bone- or tumor-seeking and increase the therapeuticpotential of the radium solution instead of being a health hazard. Itshould be a complexing agent that does not negatively affect thebone-seeking properties of radium, though. For example, can aTCMC-labeled urea derivative scavenge ²¹²Pb produced in the radiumsolution during transport and storage between the production site andthe hospitals whereby the product is going to be administered.

Although it is possible to reduce the susceptibility by addingradiolytic inhibitors, tumor targeting peptides and peptide mimetics areoften more susceptible to radiolysis and should probably be supplied ina kit format whereby they are added a few hours to a few minutes beforeadministration of ²²⁴Ra solutions with relatively long shelf-lives.

It is known in the field that calixarenes and EDTA to some extent cancomplex radium and also complex lead and bismuth. However, in thecurrent work we found chelators that would leave radium mainlyuncomplexed or weakly complexed, as determined by in vivobiodistribution measurements, while being able to rapidly and withrelevant stability, complex the longest living daughter ²¹²Pb. Theselective complexation can be used to make at least lead bone- ortumor-seeking while maintaining the favourable properties of radium interms of treating sclerotic diseases, like skeletal metastases. The²¹²Pb complex that targets bone or tumor cells generates the alphaemitter ²¹²Bi from the decay of ²¹²Pb. Thus, the beta emitter ²¹²Pb isused as an indirect alpha source for irradiating the targeted cells ortissue. Other potential chelates which could be suitable for ²²⁴Radaughter nuclide scavenging besides TCMC and DOTA includes but are notlimited to phorphyrins, DTPA and DTPA derivatives and also carboxyllinked DOTA.

Lead-212 is by far the longest living of the progenies from ²²⁴Ra andthis is the most important to complex, as it is an in vivo generator forthe short-lived alpha-emitter ²¹²Bi. If a ²¹²Pb-chelate is taken up inbone or in tumor cells ²¹²Bi will also likely be retained in the target.In a ²²⁴Ra solution in equilibrium with progenies there will be morethan 10 times of ²¹²Pb vs. ²¹²Bi atoms. Thus, the amount of radiationgenerated from the ²¹²Bi atoms in these solutions are modest andprobably not of a toxicologically importance compared with ²²⁴Ra and²¹²Bi decay series. The amount of ²¹²Bi is comparable to that of the²¹¹Pb which indirectly produces an alpha particle in the ²²³Ra seriesand this has not been of a significant problem for the registration andclinical use of ²²³Ra in equilibrium with progenies.

If, however, a high degree of chelation also of ²¹²Bi in an injectateshould be needed, it may at least in some instances be necessary to adda stabilizing agent like NaI or HI since bismuth in aqueous solutionstends to exist in a state less suitable for chelation.

When comparing with current approved alpha-pharmaceutical for treatmentof skeletal metastases, i.e., ²²³Ra, the novel solutions describedherein could give, in one of the embodiment, a product with improvedproperties for treatment of skeletal metastases since the daughternuclide can be made targetable to circulating cancer cells and, to someextent, also soft tissue metastases. This may prevent recurrence fromcancer recolonization of the skeleton due to CTC's.

Another aspect is that the shorter half-life of ²²⁴Ra vs. ²²³Ra mayactually be of some benefit as the radium is embedded in the bonematrix. Because of the high density of the bone mineral the range ofalpha-particles is strongly reduced in bone vs. soft tissues. Especiallyin rapid mineralizing areas like osseous cancer metastases, theembedment process may be of significance when using a volume-seekingalpha-pharmaceutical.

Therefore, ²²⁴Ra could improve the tumor dose since, on average, it willbe less embedded at the time of decay.

Diseases by which the novel ²¹²Pb solutions with or without ²²⁴Ra may beused include but are not limited to primary and metastatic cancers,autoimmune diseases and artherioschlerosis. The product may beadministered intravenously or locally, including intraperitoneally, orin limb perfusion settings.

The chelators used in the novel solutions may be acyclic as well ascyclic chelators and cryptands, crown ethers, porphyrins or cyclic- ornoncyclic polyphosphonates including DOTMP and EDTMP. Also abisphosphonate, e.g., pamidronate, conjugated to DOTA, TCMC or similarmay be used as scavenger in the ²²⁴Ra solution.

One may argue that the amount of ²¹²Pb in therapeutic ²²⁴Ra solution maybe moderate to modest (i.e., at equilibrium about 1.1 times that of²²⁴Ra). If one assumes similar dosing of ²²⁴Ra as is done with ²²³Ra inpatients but correct for the half-life difference, roughly 150 kBq perkg of bodyweight would be the administered dose.

At equilibrium this would translate into a ²¹²Pb-antibody conjugatedosage of 11.5 MBq in 5 litres of blood in a 70-kg patient (if ²¹²Pb isquantitatively chelated). The number of circulating tumor cells istypically less than 10 cells per ml, thus in 5 I blood there are lessthan 50 000 tumor cell in total. If only 1 in 100 000 of the injected²¹²Pb-antibody conjugate molecules binds to the tumor cells this wouldmean at least 0.0023 Bq per cell, equivalent to approximately 127 ²¹²Pbatoms bound per cell, which would be highly destructive as it has beenreported that a mean of 25 cell bound ²¹²Pb per cell would kill 90% of acell population.

Compounds

An aspect of the present invention relates to a compound X linked to achelating moiety Z is defined by the formula I:

or a pharmaceutically acceptable salt thereof,

wherein

W is a PSMA-targeting ligand;

A⁴ is a bond or a divalent linking moiety comprising 1 to 10 carbonatoms in a chain, a ring, or a combination thereof, wherein at least onecarbon atom is optionally replaced with O, —NR³—, or —C(O)—;

G is C═O, C═S, C—NH₂, or C—NR³;

R¹ is hydrogen or a carboxylic acid protecting group;

R³ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,heterocycloalkyl, aryl, alkylaryl, and heteroaryl.

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are each independently hydrogen, alkyl,alkoxyl, or R¹⁷ and R¹⁸ are each independently hydrogen, alkyl, aryl, oralkylaryl;

R¹⁹ is selected from the group consisting of alkyl, alkoxyl, halide,haloalkyl, and CN;

m is an integer from 1 to 6; and

o is an integer from 0 to 4, wherein when o is greater than 1, each R¹⁹is the same or different.

An aspect of the present invention relates to a complex according to thepresent invention, or a pharmaceutically acceptable salt thereof,wherein

A⁴ is a bond, (CH₂)_(n), —HC(O)—, —(OCH₂CH₂)_(n)—, —(HCH₂CH₂)_(n)—,—H(CO)CH₂—, —HC(O)CH₂(OCH₂CH₂)_(n)—, or —HC(O)CH₂(HCH₂CH₂)_(n)—; and

L is a bond, (CH₂)_(n), —(OCH₂CH₂)_(n)—, —(HCH₂CH₂)_(n)—, or—C(O)(CH₂)_(n)—;

wherein n is independently 1, 2, or 3.

An aspect of the present invention relates to a complex according to thepresent invention, or a pharmaceutically acceptable salt thereof,wherein A⁴ is a bond, —(OCH₂CH₂)_(n)—, or —HC(O)CH₂(OCH₂CH₂)_(n)—; and

L is a bond, or —(OCH₂CH₂)_(n)—;

wherein n is independently 1 or 2.

An aspect of the present invention relates to a complex according to thepresent invention, or a pharmaceutically acceptable salt thereof,wherein

W has the structure:

wherein R²⁰ and R²¹ are each independently an amino acid residue linkedvia an amino group thereof to the adjacent —C(O)— group.

An aspect of the present invention relates to a complex according to thepresent invention, or a pharmaceutically acceptable salt thereof,wherein

W has the structure:

wherein R is hydrogen or a carboxylic acid protecting group.

An aspect of the present invention relates to a complex according to thepresent invention, or a pharmaceutically acceptable salt thereof, havingthe structure:

or a pharmaceutically acceptable salt thereof,

wherein R¹⁷ is aryl.

An aspect of the present invention relates to a complex according to thepresent invention, or a pharmaceutically acceptable salt thereof,wherein the complex is PSMA-617:

With a radionuclide such as ²¹²Pb linked/chelated to four N.

An aspect of the present invention relates to a complex according to thepresent invention, wherein the DOTA unit, such as p-SCN-Bz-DOTA, issubstituted with a TCMC unit, such as p-SCN-Bz-TCMC.

The linker in PSMA-617 may also be covalently linked to from a C-atom inthe backbone instead of link to N as can be seen in the above figure.

Thus, the urea derivative may have backbone C-linked or N-linked DOTA orTCMC conjugation.

For a backbone-C linked p-SCN-Bn-DOTA or p-SCN-Bn-TCMC conjugation isthe compound:

Wherein Z is:

And wherein X is: —OH or NH₂.

X is —OH for p-SCN-Bn-DOTA and X is NH₂ for p-SCN-Bn-TCMC.

This means that the formula for backbone-C linked p-SCN-Bn-DOTA orp-SCN-Bn-TCMC will be:

wherein X is: —OH or NH₂.

Backbone-C linked p-SCN-Bn-DOTA is:

p-SCN-Bn-DOTA-PSMA ligand 2

Thus, an aspect of the present invention related to a compound which isp-SCN-Bn-DOTA-PSMA ligand 2.

Backbone-C linked p-SCN-Bn-TCMC is:

p-SCN-Bn-TCMC-PSMA ligand 1

Thus, an aspect of the present invention related to a compound which isp-SCN-Bn-TCMA-PSMA ligand 1 or p-SCN-Bn-DOTA-PSMA ligand 2 as disclosedabove, and the examples.

The p-SCN-Bn-TCMC-PSMA ligand (ligand 1) and p-SCN-Bn-DOTA-PSMA ligand(ligand 2) (as shown in the examples) is linked via the carbon backbone(C-backbone) to the rest of the compound and has an extended linkerregion including a isothiocyanato-benzyl linker and also uses a carbonsubstituted chelator with all 4 chelator arms free as opposed toPSMA-617 which has a shorter linker region and uses one of the chelatorarms as linker attachment. It is shown in the later examples herein thatthese differences cause a significant different biodistribution of theradiolabelled product, making it more suitable for targeting of ²¹²Pb toPSMA-expressing tumors, as it reduces kidney exposure compared toPSMA-617.

In one embodiment of the present invention can the compounds of thepresent invention, such as p-SCN-Bn-TCMC-PSMA ligand 1 andp-SCN-Bn-DOTA-PSMA ligand 2, be in the form of a trifluoroacetic acidsalt.

CTT1401 and CTT1403 and derivatives (Choy et al, 2017) with TCMC or DOTAvariants may also be used with ²¹²Pb in the complexes of the presentinvention.

By preparing a PSMA binding Urea derivative according to the presentinvention with a readily accessible amino group or another groupsuitable for conjugation, p-SCN-Bn-TCMC can be conjugated to the PSMAbinding compound. Subsequent purification may be needed beforeradiolabelling with ²¹²Pb with or without ²²⁴Ra in solution.

Thus, in one embodiment of the present invention is the linkerp-SCN-Bn-TCMC. One embodiment of the present invention relates to acomplex according to the present invention, wherein the linker-chelatoris p-SCN-Bn-TCMC. One embodiment of the present invention relates to acomplex according to the present invention, wherein the linker isp-SCN-Bn-DOTA. In other words -p-SCN-Bn- is the end part of the linkerregion which is attached to the TCMC or DOTA chelator group via thecarbon backbone.

Human serum albumin can be used to prolong the half-life of drugs. Thus,in an embodiment the ²¹²Pb-labeled PSMA binding urea derivativeaccording to the present invention further comprises a group that canassociate with albumin to increase the circulation half-life of theradiolabeled product.

A further embodiment of the present invention relates to a complexaccording to the present invention, wherein ²¹²Pb-labeled PSMA bindingurea derivative according to the present invention further compriseshuman serum albumin that has been directly conjugated to the complex, oris associated to the complex, for example through liposomes.

Pharmaceutical Compositions

An embodiment of the present invention relates to a solution comprisingthe compound and/or the complex of the invention. The solution can alsobe a pharmaceutical composition.

Usually is an important element of a pharmaceutical composition a buffersolution, which to a substantial degree maintain the chemical integrityof the radioligand and is being physiologically acceptable for infusioninto patients.

In one embodiment of the present invention, the pharmaceuticalcomposition comprises one or more pharmaceutically acceptable carriersand/or adjuvants.

Acceptable pharmaceutical carriers include but are not limited tonon-toxic buffers, fillers, isotonic solutions, etc. More specifically,the pharmaceutical carrier can be but are not limited to normal saline(0.9%), half-normal saline, Ringer's lactate, 5% Dextrose, 3.3%Dextrose/0.3% Saline. The physiologically acceptable carrier can containan anti-radiolytic stabilizer, e.g., ascorbic acid, which protect theintegrity of the radiopharmaceutical during storage and shipment.

An aspect of the present invention relates to a pharmaceuticalcomposition comprising the complex according to the present invention,and a diluent, carrier, surfactant, and/or excipient.

An aspect of the present invention relates to single agent ²¹²Pb-labeledligand. This would be the compound of the present invention complexedwith ²¹²Pb and without any further radionuclides, such as ²²⁴Ra.

An aspect of the present invention relates to a dual targeting solutioncontaining ²¹²Pb-labeled ligand and cationic or weakly complexed ²²⁴Ra.This would be the compound of the present invention complexed with ²¹²Pbwith ²²⁴Ra present as cationic or weakly complexed ²²⁴Ra.

An aspect of the present invention relates to a pharmaceuticalcomposition comprising the compound and/or the complex according to thepresent invention, further comprising ²²⁴Ra. ²²⁴Ra can be cationic. Theaddition of ²²⁴Ra allows for dual targeting for example when presenttogether with ²¹²Pb.

An aspect of the present invention relates to a pharmaceuticalcomposition comprising the complex according to the present invention,wherein the radioactivity is 100 kBq to 100 MBq per dose.

An aspect of the present invention relates to a pharmaceuticalcomposition comprising the complex according to the present invention,wherein the amount of ²²⁴Ra and ²¹²Pb is in radioactive equilibrium.

An aspect of the present invention relates to a pharmaceuticalcomposition comprising the complex according to the present invention,wherein the activity ratio (MBq) between ²¹²Pb to ²²⁴Ra is between 0.5and 2, such as 0.8-1.5, or such as 0.8-1.3, or preferably such as0.9-1.15.

Kits

The solution should be made physiologically suitable for injectionseither at a centralized production site or be made up by a kit system oftypically 2-4 vials whereby being physiologically suitable for injectionafter combination of the kit vials.

An aspect of the present invention relates to a kit comprising a firstvial comprising a radiopharmaceutical composition according to thepresent invention, and a second vial comprising a neutralizing solutionto adjust pH and/or isotonicity of the radiopharmaceutical compositionprior to administration to a patient.

For, e.g., a monoclonal antibody, it is usually advisable be keep theself-dose of the alpha particle producing radiopharmaceutical solutionbelow 0.5 kGy to avoid reduced binding properties due to radiolysis.Thus, a kit system whereby chelator conjugated ligand is added to the²²⁴Ra (including daughters) solution a few hours to 10 minutes beforeinjection is advised for concentrated solutions intended for remoteshipping, depending of the radiolytic resistance of the radioligand thatis generated.

An aspect of the present invention relates to a kit comprising a firstvial comprising a ²²⁴Ra solution; a second vial comprising a complexingagent selected from the group consisting of p-SCN-Bn-DOTA-PSMA ligand,p-SCN-Bn-TCMC-PSMA ligand, acyclic chelators, cyclic chelators,cryptands, crown ethers, porphyrins or cyclic or noncyclicpolyphosphonates, DOTMP, EDTMP, bisphosphonate derivatives, DOTA, a DOTAderivative, pamidronate conjugated to DOTA, TCMC, a TCMC derivative,pamidronate conjugated to TCMC, antibody-conjugated-DOTA,antibody-conjugated-TCMC, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO,DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A, wherein the complexing agentis capable of complexing a daughter nuclide of ²²⁴Ra such as ²¹²Pb, andwherein the complexing agent does not complex ²²⁴Ra in thepharmaceutical solution; and optionally, instructions for mixing thefirst vial and the second vial, thereby forming a pharmaceuticalcomposition ready to be administered to a patient 1 minute to 12 hoursafter mixing.

In one embodiment of the present invention is the kit for use as amedicament. In a specific embodiment, the term “²²⁴Ra solution” is to beunderstood as ²²⁴Ra is free in the solution and not coupled to e.g. asurface such as a resin.

In an embodiment, the kit comprises a third vial comprising aneutralizing solution to adjust pH and/or isotonicity of theradiopharmaceutical solution prior to administration to a patient.

In yet a preferred embodiment, the amount of ²²⁴Ra and ²¹²Pb is inradioactive equilibrium in the first vial.

In yet another preferred embodiment the activity ratio (MBq) between²¹²Pb to ²²⁴Ra in the first vial is between 0.5 and 2, such as 0.8-1.5,or such as 0.8-1.3, or such as 0.9-1.15.

In yet another embodiment the first vial has a radioactivity in therange 100 kBq to 100 MBq.

In one embodiment of the present invention, the chelator conjugatedligand is added to the ²²⁴Ra (including daughters) solution 30 min to 5hour before injection, such as 1-3 hours before injection.

In one embodiment of the present invention, the chelator conjugatedligand is added to the ²²⁴Ra (including daughters) solution 1 min to 20min before injection.

In one embodiment of the present invention, the chelator conjugatedligand is added to the ²²⁴Ra (including daughters) solution 1 min to 10min before injection.

A kit with a chelate labelled protein or peptide in one vial and a ²²⁴Rasolution in another vial whereby the content of the two are mixed 12hours to 1 minute before administration also forms part of theinvention. In an embodiment, the mixing takes place a few hours (such as5) to 30 minutes before administration to a patient as to bind ²¹²Pb andor ²¹²Bi to the chelate.

In one embodiment of the present invention, the content of the two aremixed 30 min to 1 hour before injection.

In one embodiment of the present invention, the content of the two aremixed 1 min to 20 min before injection.

In embodiment of the present invention, the content of the two are mixed1 min to 10 min before injection.

Optionally, a third vial containing a liquid used for dilution andisotonicity adjustment before administration of the radiopharmaceuticalsolution could be used. This third vial may contain EDTMP, which couldchelate ²¹²Bi, if needed.

Medical Applications

An aspect of the present invention relates to the radiopharmaceuticalcomposition according to the present invention for use as a medicament.

In one embodiment of the present invention is the disease cancer.

An aspect of the present invention relates to the radiopharmaceuticalcomposition according to the present invention for use in the treatmentof soft tissue and/or skeletal disease. The treatment is focused onPSMA-expressing disease including soft tissue- and skeletal disease.

In one embodiment of the present invention is the skeletal diseaseselected from the group consisting of soft tissue and or skeletalmetastases from cancers to the breast, prostate, kidneys, lung, bone, ormultiple myeloma.

In one embodiment of the present invention is the cancer prostatecancer. The cancer can also be breast cancer. The cancer can be kidneycancer. The cancer can also be lung cancer. The cancer can also be bonecancer. The cancer can also be multiple myeloma. The cancer can bemetastases from these types of cancer.

In one embodiment of the present invention is the solution administeredat a dose in the range 50-150 kBq per kg of bodyweight, such as 50-100kBq per kg of bodyweight.

An aspect of the present invention relates to a method of treatment ofmalignant or non-malignant disease by administration of aradiopharmaceutical composition according to the present invention to anindividual in need thereof.

This may be used with purified ²¹²Pb-labeled ligand or in a dualtargeting solution whereby ²²⁴Ra will act as a skeletal treatment, and²¹²Pb-urea-deriviative will act as a systemic therapy against cells theexpressing PSMA antigen which is associated with advances metastaticprostate cancer.

Thus, the complexes and the solutions of the present invention can beused in the treatment of metastatic prostate cancer.

Another embodiment of the present invention relates to a pharmaceuticalsolution with dual targeting properties whereby ²¹²Pb is complexed by aurea based PSMA targeting agent as disclosed herein, and cationic ²²⁴Rais targeting bone metastases though calcium mimetic bone incorporation.

Methods for Preparation

An aspect of the present invention relates to a method for providing aradiopharmaceutical composition according to the present invention, themethod comprising providing a first solution wherein the amount of ²²⁴Raand ²¹²Pb is in radioactive equilibrium; providing a second solutioncomprising a complexing agent that is selected from the group consistingof p-SCN-Bn-DOTA-PSMA ligand, p-SCN-Bn-TCMC-PSMA ligand, acyclicchelators, cyclic chelators, cryptands, crown ethers, porphyrins orcyclic or noncyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate,DOTA, a DOTA derivative, pamidronate conjugated to DOTA, TCMC, a TCMCderivative, pamidronate conjugated to TCMC, antibody-conjugated-DOTA,antibody-conjugated-TCMC, HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO,DTPA, CHX-DTPA, AAZTA, DEDPA, and oxo-Do3A, wherein the complexing agentis capable of complexing a daughter nuclide of ²²⁴Ra, such as ²¹²Pb, andwherein the complexing agent does not complex ²²⁴Ra; and mixing thefirst composition and the second composition, thereby providing apharmaceutical composition according to the present invention.

PSMA Derivatives

PSMA (also named Prostate Specific Cancer Antigen, PSM, FGCP, FOLH,GCP2, mGCP, GCPII, NAALAD1, NAALAdase, FOLH1, Glutamate carboxypeptidase2, Glutamate carboxypeptidase II, Membrane glutamate carboxypeptidase,N-acetylated-alpha-linked acidic dipeptidase I,Pteroylpoly-gamma-glutamate carboxypeptidase, Folylpoly-gamma-glutamatecarboxypeptidase, Folate hydrolase 1, Prostate-specific membraneantigen, Cell growth-inhibiting protein 27) is a prostate epithelialcell membrane antigen of type II transmembrane protein and consist of ashort NH2-terminal cytoplasmic domain, hydrophobic transmembrane region,and a large extracellular domain. PSMA is an enzyme that in humans isencoded by the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750amino acids and weighs approximately 84 kDa.

Expression of PSMA is restricted to a few healthy tissues such aslacrimal and salivary glands, proximal renal tubules, epididymis, ovary,the luminal side of the ileum-jejunum and astrocytes within the centralnervous system (CNS); healthy prostate gland expresses comparativelylittle PSMA, which is confined within the apical epithelium of secretoryducts. In these non-malignant tissues, uptake of PSMA-targeted probesmay be limited by an intact blood-brain barrier, a healthy proximalsmall bowel lumen, and truncated cytoplasmic expression of PSMA withinnormal prostate. PSMA is most associated with high grade androgenindependent, metastatic disease, although PSMA is expressed in mostprimary prostate tumors regardless of androgen status.

Small molecular PSMA inhibitors are zinc binding compounds and can beclassified into three types 1) phosphonate-, phosphate-, andphosphoramidate compounds; 2) thiols; and 3) ureas. The urea derivativesseem to have particularly interesting properties as carrier forradionuclides for diagnosis and therapy.

The current invention relates to use of ²¹²Pb-urea derivatives. It maybe combined with androgen deprivation therapy to enhance PSMA expressionfor better uptake of radioligand (Bakht et al, 2017).

Activity Level

If a ²¹²Pb-labeled urea derivative is used alone, the activity levelwould typically be between 1 MBq and 500 MBq per patient, more typically10-100 MBq per patient.

If ²²⁴Ra in equilibrium with ²¹²Pb complexed with a PSMA binding ureaderivative the dosing would typically be between 0.1 MBq and 100 MBq perpatient, more typically between 1 and 20 MBq per patient.

In a special embodiment an urea derivative based PSMA targeting complexaccording to the present invention, labeled with ²²⁷Th, which producesbone-seeking ²²³Ra for dual targeting.

The ²²⁷Th may be pure or containing various amounts of ²²³Ra eg. 10%,50%, 100% or 250% of ²²³Ra compared to ²²⁷Th.

General

It should be understood that any feature and/or aspect discussed abovein connections with the compounds according to the invention apply byanalogy to the methods described herein.

The following figures and examples are provided below to illustrate thepresent invention. They are intended to be illustrative and are not tobe construed as limiting in any way.

EXAMPLES

In the following examples the xenograft model used is a tumor model withintermediary level of PSMA-ligand uptake of typically 10-15% of injecteddose per gram (% ID/g) in mouse xenografts with ¹⁷⁷Lu-PSMA-617 asopposes to the PC3 PIP model used by other researchers which shows anuptake of typically 30-40% ID/g in mouse xenografts.

Example 1. Novel PSMA-Binding Chelator Ligands Compared with PSMA-617

Background: Several carrier molecules for radioligand targeting ofprostate specific membrane antigen (PSMA) exists. Lutetium-177 labeledPSMA-617 (¹⁷⁷Lu-PSMA-617) is the compound in most advanced clinicaldevelopment stage for use in radionuclide therapy. This molecule workswell and give relevant tumor to normal tissue ratios for longer lived(i.e., a half-life of a few days) radionuclides, including ¹⁷⁷Lu and²²⁵Ac, but at early times points (typically a few hours after injection)shows high uptake in kidneys. With shorter lived radionuclides like²¹²Pb (half-life of 10.6 hours), the initial kidney uptake represents apotential toxicity problem. It is therefore advantageous to use aPSMA-ligand with less kidney uptake, but this should not compromise thetumor uptake. The PSMA ligand molecules are made up of (1) aPSMA-binding region, (2) a linker region and (3) a chelator, whereby thelinker region connects the (1) and (3). The linker region also is usedto adjust molecular size and polarity to affect the in vivo distributionproperties. The PSMA-binding region (motif) used in PSMA-617 is astructure that can be found in several molecules of this class developedby several different inventors and researchers including PSMA-11 andPSMA I&T as well as ¹³¹I- and ²¹¹At-labelled PSMA binding ligands.

As can be seen, the p-SCN-Bn-TCMC-PSMA ligand 1 has an extended linkerregion including a isothiocyanato-benzyl linker and also uses a carbonsubstituted chelator with all 4 chelator arms free as opposed toPSMA-617 which has a shorter linker region and uses one of the chelatorarms as linker attachment. It is shown in later examples herein thatthese differences cause a significant different biodistribution of theradiolabelled product, making p-SCN-Bn-TCMC-PSMA ligand 1 it moresuitable for targeting of ²¹²Pb to PSMA-expressing tumors, as it reduceskidney exposure compared to PSMA-617.

Materials and methods: The PSMA-ligand precursors for radiolabeling weresynthesized by a subcontractor commercial synthesis laboratory.

PSMA-617 was synthesized according to procedures described in theliterature. The p-SCN-Bn-TCMC precursors were synthesized according toprocedures described in the literature. In the final synthesis step theTCMC-PSMA ligand was synthesized by conjugating p-SCN-Bn-TCMC to aminogroup of a PSMA binding ligand intermediate. PSMA-617 andTCMC-Bn-PSMA-ligand 1 were both purified by HPLC to a purity of >98% anddried and stored as the trifluoroacetic acid salts. Structures andmolecule weights were determined with ¹H-NMR and MS analysis.

The p-SCN-Bn-TCMC-PSMA ligand 1 trifluoracetic acid salt has thechemical formula C₆₅H₈₆F₁₂N₁₄O₂₁S and a molecular weight of 1659.52g/mol.

Chemical Name (IUPAC):(((1S)-1-carboxy-5-((2S)-3-(naphthalen-2-yl)-2-((1r,4S)-4-((3-(4-((1,4,7,10-tetrakis(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)methyl)cyclohexane-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamicacid; trifloroacetic acid (1:4)

The PSMA-617 trifluoroacetic acid salt has the chemical formulaC₅₇H₇₅F₁₂N₉O₂₄ and a molecular weight of 1498.25 g/mol.

p-SCN-Bn-TCMC-PSMA ligand 1 trifluoracetic acid salt.

PSMA-617 trifluoroacetic acid salt

As can be seen, the p-SCN-Bn-TCMC-PSMA ligand 1 has an extended linkerregion including a benzyl linker and also uses a carbon substitutedchelator with all 4 chelator arms free as opposed to PSMA-617 which hasa shorter linker region and uses one of the chelator arms as linkerattachment. It is shown in the following examples that these chemicaldifferences cause a significant different biodistribution of theradiolabelled product, making it more suitable for targeting of ²¹²Pb toPSMA-expressing tumors, as it reduces kidney exposure compared toPSMA-617.

In conclusion, a novel molecule with same PSMA-binding region, but withdifferent linker region and different chelation properties compared withPSMA-617 is described.

Example 2

A carbon substituted p-SCN-Bn-DOTA-PSMA ligand 2 for radiolabeling with²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, ²²⁷Th, ¹⁷⁷Lu, etc.

Compared to PSMA-617 the ligand has larger size and a p-SCN-Bn-DOTAgroup, i.e., both a different linker region and a differentDOTA-chelator version where all four chelator arms are free to causechelation of radionuclides as compared to PSMA-617.

The p-SCN-Bn-DOTA-PSMA ligand 2 is the DOTA analogue ofp-SCN-Bn-TCMC-PSMA ligand 1 described in example 1 and has carbon linkerto the chelator backbone leaving the chelator groups free to interactwith the radiolabel and is therefore expected to improve chelatestability after radionuclide labelling compared with radiolabelledPSMA-617. Due to the additional lipophilic benzyl unit in the linkerregion and larger size of molecule, less kidney uptake is expectedcompared with PSMA-617. This molecule can be synthesized in the samefashion as p-SCN-Bn-TCMC-PSMA ligand 1 by using a DOTA based precursorinstead of a TCMC-based precursor in the last step of synthesis.

p-SCN-Bn-DOTA-PSMA ligand 2 trifluoracetic acid salt.

The p-SCN-Bn-DOTA-PSMA ligand 2 trifluoracetic acid salt has thechemical formula C₆₅H₈₂F₁₂N₁₀O₂₅S and a molecular weight of 1663.46g/mol.

With the carbons substituted attachment to the DOTA, this molecule hasproperties very suitable for radiolabeling with ²¹²Pb, ²¹²Bi, ²¹³Bi,²²⁵Ac, ²²⁷Th, and also for positron emission tomography (PET) compatibleradionuclides like, ⁶⁸Ga, as well.

Thus, a novel PSMA-binding molecule is described, p-SCN-Bn-DOTA-PSMAligand 2, suitable for radioligand imaging and therapy.

In conclusion, carbon substituted p-SCN-Bn-DOTA-PSMA ligand 2 isdescribed with different properties in terms of size and chelationproperties compared with PSMA-617.

Example 3. Radionuclides Tested

Lutetium-177 was purchased as ready to use ¹⁷⁷LuCl₃ dissolved in dilutedHCl. Lead-212 was obtained from ²²⁴Ra based solutions. Radium-224 wasmade from ²²⁸Th bound to Actinide resin (Eichrom Technologies, LLC) byeluting a column containing actinide resin with immobilized ²²⁸Th with 1M HCl. The eluate was purified on a second Ac-resin column and theeluate evaporated to dryness using an evaporation vial with a cap withgas inlet and outlet placed in a heater block at approximately 110° C.and a gentle stream of nitrogen gas to evaporate of the solvent. Whenthe evaporation vial was empty from solvent it was added 0.1 M HCl todissolve the residue, typically 200-400 μl.

Example 4. Radiolabeling of PSMA-Binding Ligands

In general, ¹⁷⁷Lu and ²²⁴Ra/²¹²Pb solutions were adjusted with HCl with10% 5 M ammonium acetate to the desired volume and pH 5-6. ThePSMA-binding ligands were dissolved in 0.1 M HCl with 0.5 M ammoniumacetate to a concentration of 1 mg/ml. Typically a concentration of 20microgram per 1 ml radioactive solution were used. The reaction mixturewas incubated on a shaker for 15-30 minutes, typically a labelling wasevaluated by thin layer chromatography. Lead-212 labeling was performedat room temperature or 37° C. with the p-SCN-Bn-TCMC-PSMA ligand 1,while ¹⁷⁷Lu-labeling and ²¹²Pb-labeling of PSMA-617 was performed at 90°C. Typical radiolabeling yields were in the range 90-100% with thecompounds and the radionuclides tested, providing a concentration of 1μg per 20 μl or higher were used. In conclusion, radiolabeling of bothligands worked well. The novel p-SCN-Bn-TCMC-PSMA ligand 1 could beradiolabelled with ²¹²Pb at room temperature as opposed to¹⁷⁷Lu-PSMA-617 that had to be labeled at elevated temperature.

Example 5: Thin Layer Chromatography Analyses

Thin layer chromatography (TLC) was performed using chromatographystrips (model #150-772, Biodex Medical Systems Inc, Shirley, N.Y., USA).A formulation buffer (FB) consisting of 7.5% human serum albumin and 5mM EDTA in DPBS and adjusted to approximately pH 7 with NaOH was mixedwith the antibody conjugates in ratio 2:1 for at least 5 minutes beforeapplication to the strips to determine free radionuclide. A small beakerwith about 0.5 ml of 0.9% NaCl was used to place strips with a samplespot in. To the strip was typically added 1-4 μl of sample atapproximately 10% above the bottom of the strip. After the solvent fronthad moved to about 20% from the top of the strip, the strip was cut inhalf and each half was placed in a 5 ml test tube for counting. In thissystem radiolabeled ligand stays at the bottom half while radionuclidecomplexed with EDTA migrates to the upper half. It was verified thatfree cations of both ²¹²Pb and ¹⁷⁷Lu would complex with EDTA and movedto the top.

In conclusion, a TLC system was used allowing a rapid determination ofradiolabeling yield that would distinguish effectively betweenradioligand and free radionuclide.

Example 6. Separation of ²¹²Pb from ²²⁴Ra Solutions

Radiolabeled ligand can be used as a component in ²²⁴Ra dual targetingsolutions whereby ²²⁴Ra targets skeletal disease and ligand targetssystemic metastatic disease. Alternatively, ²²⁴Ra generator solutionscan be used for producing ²¹²Pb-labeled ligand by labelling in situ,i.e., ²¹²Pb is complexed by the ligand in the presence of ²²⁴Ra. To 40μl Radium-224/²¹²Pb solutions in 0.5 M ammonium acetate, pH 5-6 wasadded 2 μl (1 μg/μl) of either p-SCN-Bn-TCMC-PSMA ligand 1 or PSMA-617and reacted as described to generate the ²¹²Pb-labeled ligands. Topurify ²¹²Pb-PSMA ligand the product was added about 10 μl of aformulation buffer consisting of 7% bovine serum albumin, 10 mM EDTA,and 10 mg/ml ascorbic acid. Thereafter the reaction mixture was added toa Sephadex G-10 column PD MiniTrap G-10 (GE Healthcare Life Sciences)and eluted with 0.9% NaCl. The eluate containing the ²¹²Pb, typicallythe fractions eluted after application of 0.7-1.5 ml, was collected andanalysed on a gamma counter and TLC and radioligand binding assay wasperformed. The product purification procedure had high radiochemicalyield (typically >80%) and had a high radiochemical purity with ²²⁴Raamounting to less than 0.4% compared with ²¹²Pb-labeled ligand,typically.

In conclusion, p-SCN-Bn-TCMC-PSMA ligand 1 and PSMA-617 could beradiolabelled with ²¹²Pb in the presence of ²²⁴Ra yielding a dualtargeting solution for combined bone metastases targeting with ²²⁴Ra andsystemic tumor cell targeting with ²¹²Pb-labeled PSMA ligand.

It was also found that using Sephadex G-10 gel filtration column, both²¹²Pb-labeled PSMA-ligands tested could be separated from ²²⁴Ra in thesolution with a recovery above 80% and breakthrough of ²²⁴Ra of lessthan 0.4% yielding a highly purified ²¹²Pb-labeled PSMA radioligand forstand-alone PSMA targeting.

Example 7. Stability Testing of ²¹²Pb-Labeled Ligands In Vitro

Radiolabeled ligand in ²²⁴Ra/²¹²Pb solution was mixed 1:1 with PBS orbovine serum albumin and incubated at 37° C. for up to 48 hours. TLCanalyses were performed at 1 h, 4 h, 24 h and 48 h of incubation.

The data are shown in table 1 and indicates that ²¹²Pb continuously,after it is generated from ²²⁴Ra, react with the ligand and maintain ahigh percentage of radiochemical purity even after 48 hours. Thus, thePSMA-ligands are compatible with the use of a ²²⁴Ra solution for in situproduction of radioligand that is suitable for centralized productionand shipment to the end user.

TABLE 1 Radiochemical purity of ²¹²Pb-TCMC-PSMA ligand 1 and²¹²Pb-PSMA-617 in PBS and FBS ²¹²Pb-p-SCN-Bn- Time since start TCMC-PSMAligand 1 ²¹²Pb-PSMA-617 of incubation PBS FBS PBS FBS 1 h 93.26% 93.50%91.62% 92.13% 4 h 95.04% 94.18% 94.33% 94.37% 24 h 95.00% 93.54% 93.26%94.54% 48 h 95.11% 90.81% 93.17% 95.65%

Conclusion: The data indicate that p-SCN-Bn-TCMC-PSMA ligand 1 andPSMA-617 labelled with ²¹²Pb are stable in ²²⁴Ra solutions for aprolonged period of time indicating compatibility with centralizedproduction and shipment to the end user of ready to use product. Suchsolutions could be used for treatment against cancer.

Example 8. Prostate Cancer Cell Binding of p-SCN-Bn-TCMC-PSMA Ligand 1and PSMA-617 Labeled with ²¹²Pb

the cell binding fractions measured by adding about 1 ng of radioligandto 0.2 ml of C4-2 cells (5×10⁷ cells per ml) in a 5 ml test tube andincubate for 1 h before measuring applied activity, wash cells 3 timeswith 0.5 ml 0.5% bovine serum albumin in DPBS and thereafter recount thewashed cell pellet. From multiple experiments it was found that the %bound was in the range of 40-53% after subtracting for nonspecificbinding. Nonspecific binding was measured by blocking the cells withexcess 10 μg/ml of unlabelled ligand before adding radioligand. Nosignificant difference in cell-binding was found between radiolabelledp-SCN-Bn-TCMC-PSMA ligand 1 and PSMA-617. In conclusion, the radioligandof radiolabelled p-SCN-Bn-TCMC-PSMA ligand 1 and PSMA-617 had similarcell binding properties in vitro indicating that p-SCN-Bn-TCMC-PSMAligand 1 has relevant antigen binding ability.

Example 9. Radiolytic Stability of p-SCN-Bn-TCMC-PSMA Ligand 1 Assessedby Radioligand Binding Ability with Prostate Cancer Cells and TLCAnalyses

Table 2 shows the cell binding fractions measured by adding about 1 ngof radioligand to 0.2 ml of C4-2 cells (5×10⁷ cells per ml) in a 5 mltest tube and incubate for 1 h before measuring applied activity, washcells 3 times with 0.5 ml 0.5% bovine serum albumin in DPBS andthereafter recount the washed cell pellet. Initial activity in thesolution was about 5 MBq/ml causing an absorbed radiation dose to thesolution of about 1.8 Gy after 24 h and 3.3 Gy after 48 hours. The data(Table 2) indicate a slight decline at the longest exposure time but ingeneral a relatively strong radiolytic resistance of the ligand wasobserved, which is compatible with centralized production and shipmentto the end user ²²⁴Ra-based generator solution with or without removalof ²²⁴Ra prior to the administration of product.

TABLE 2 Binding ability of ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 when keptin ²²⁴Ra solution. Time since start Cell binding TLC measured ofincubation fraction RCP 0.5 h 93.81% 1 h 92.84% 4 h 52.3% 94.75% 24 h48.16% 94.85% 48 h 37.18% 94.85%

Conclusion: The data indicate that ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 isstable in ²²⁴Ra solutions for a prolonged period of time and thePSMA-ligand is capable of complexing ²¹²Pb as it is generated from ²²⁴Raduring storage, and provided that the absorbed radiation dose is keptbelow about 2 kGy such solutions could be used for treatment againstcancer.

Example 10. Biodistribution of Radiolabelled Ligands in Nude Mice withC4-2 PSMA-Positive Xenografts

The biodistribution of ²²⁴Ra/²¹²Pb solutions with p-SCN-Bn-TCMC-PSMAligand 1 and PSMA-617 labeled with ²¹²Pb and ¹⁷⁷Lu were compared afterintravenous injection in nude mice with C4-2 xenografts at various timepoints after injection. Each group usually consisted of three mice. The²¹²Pb labelling was above 92% for the products. The molar concentrationof ligand was significantly lower for the ¹⁷⁷Lu-PSMA-617 since muchhigher level of radionuclide was used with ¹⁷⁷Lu vs. ²¹²Pb.Approximately 16 kBq of ²²⁴Ra/²¹²Pb was injected in each animal, i.e.,approximately 0.2 nmol of ligand per mouse. Animals were givenanaesthesia and sacrificed by cervical dislocation followed bydissection and harvesting of tissue-, blood- and urine samples. Thesamples were weighed and counted on a gamma counter.

Example 11. Comparison of Tumor Binding and Kidney Uptake ofp-SCN-Bn-TCMC-PSMA Ligand 1 and PSMA-617 Labelled with ²¹²Pb in Mice

The biodistribution of ²²⁴Ra/²¹²Pb solutions with p-SCN-Bn-TCMC-PSMAligand 1 and PSMA-617 labeled with ²¹²Pb were compared after intravenousinjection in nude mice with C4-2 xenografts at 4 h after injection.Three mice in each group. The ²¹²Pb labelling was above 92% for bothproducts. The molar concentration was the same for both products, i.e.,12.5 nmol per MBq. Approximately 16 kBq of ²²⁴Ra/²¹²Pb was injected ineach animal, i.e., approximately 0.2 nmol of ligand per mouse. Animalswere given anaesthesia and sacrificed by cervical dislocation followedby dissection and harvesting of tissue-, blood- and urine samples. Thesamples were weighed and counted on a gamma counter. Results: Four hoursafter injection, the ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 to PSMA-617ratios were as follows: tumor 1.35; kidneys 0.20; blood 1.21; liver2.67; spleen 0.71. It was confirmed by counting samples after 3 daysstorage that the ²²⁴Ra biodistribution was not significantly altered byeither of the PSMA-directed ligands. Discussion: ²¹²Pbp-SCN-Bn-TCMC-PSMA ligand 1 showed a significant differentbiodistribution compared to ²¹²Pb-PSMA-617 and especially noteworthy isthe very low, and favourable ratio for uptake in the kidneys as kidneysare expected to be the main dose limiting normal tissue related to PSMAradioligand therapy with relatively short-lived alpha-emitters (FIG. 1).In conclusion, ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 shows very promisingearly time point biodistribution compared with PSMA-617 which isimportant when using shorter lived radionuclides like ²¹²Pb.

Example 12. Comparison of Tumor Binding and Kidney Uptake of²¹²Pb-Labeled p-SCN-Bn-TCMC-PSMA Ligand 1 and ¹⁷⁷Lu-Labeled PSMA-617 inMice

Methods: Tumor and kidney uptake of ²¹²Pb-labeled p-SCN-Bn-TCMC-PSMAligand 1 and ¹⁷⁷Lu-labeled PSMA-617 was compared at 1 hours and 4 hoursafter administration of the products by intravenous injection into nudemice with C4-2 xenografts as described in Example 9. Results: The tumorsand the kidneys were the tissues taking up the largest amounts ofradioactivity. As examples the tumor and kidney uptake of²¹²Pb-p-SCN-Bn-TCMC-PSMA were on average 13.9 and 8.1 percent ofinjected dose per gram (% ID/g) respectively at 4 hours after injection.The tumor and kidney uptake of ¹⁷⁷Lu-PSMA-617 were on average 13.6 and17.4% ID/g respectively 4 hours after injection. It is noteworthy thatthe molar amount of ligand injected was much lower for PSMA-617, whichis known to reduce kidney uptake, but still the novel ²¹²Pb-labeledcompound showed less kidney uptake. The tumor-to-kidney ratios at the4-hour timepoint were as follows: ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1,1.7; ¹⁷⁷Lu-PSMA-617, 0.8.

The average tumor-to-kidney ratios determined at the 1-hour point afteradministration were for ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1, 0.40 and for¹⁷⁷Lu-PSMA-617, 0.17. In conclusion, despite a higher molar ligandconcentration for ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1, it showed bettertumor to kidney ratios than ¹⁷⁷Lu-PSMA-617 indicating that it may bewell suited for ²¹²Pb based alpha emitter radioligand therapy.

Example 13. Biodistribution of Single Targeting Solution Containing²¹²Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 in Mice with PSMA-Positive Xenografts

Using a ²¹²Pb/²²⁴Ra solution for reaction with p-SCN-Bn-TCMC-PSMA ligand1 as described, purified ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 was purifiedusing a Sephadex G-10 gel filtration column and about 30 kBq and 300 ngof the purified radioligand product was injected per animal. The dataare shown in Table 3. As can be seen the kidney activity is reducedrelatively quickly, while the tumor uptake shows good retention. Thetumor-to-tissue ratios (Table 4) indicates suitability for radioligandtargeting with ²¹²Pb.

In conclusion, ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 shows relevanttargeting properties for use in radioligand therapy againstPSMA-expressing prostate cancer.

TABLE 3 Biodistribution for ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 at varioustimepoints post injection % ID/g Organ 1 h 2 h 4 h 8 h Blood 1.77 0.540.46 0.13 Urine 253.36 250.85 32.12 6.76 Testes 4.41 0.56 0.38 0.12Prostate 13.95 3.80 1.48 −0.10 Salivary gland 0.72 0.32 0.34 0.15 Tumor26.44 15.87 14.14 14.65 Skin 1.70 0.47 0.42 0.23 Kidneys 63.96 25.419.19 4.07 Liver 2.39 1.33 1.55 1.25 Spleen 1.24 0.44 0.54 0.36 Smallintestine 0.34 0.23 0.30 0.08 Large intestine 0.25 0.39 0.18 0.17Stomach 0.16 0.22 0.10 0.09 Lungs 1.19 0.40 0.75 0.17 Heart 0.78 0.200.52 0.09 Bladder 36.86 6.85 3.53 0.26 Femur 0.87 0.40 0.57 0.74 Muscle0.62 0.22 0.23 0.04 Brain 0.61 0.04 0.07 0.02 Skull 0.77 0.38 0.37 0.63

TABLE 4 Tumor to tissue ratios for ²¹²Pb-p-SCN-Bn-TCMC- PSMA ligand 1 atvarious timepoints 1 h 2 h 4 h 8 h Tumor/blood 14.9 29.4 30.7 112.7Tumor/kidneys 0.41 0.62 1.54 3.60 Tumor/muscle 42.6 72.1 61.5 366.2Tumor/Femur 30.4 39.7 24.8 19.8

Example 14—Dosage

The radiation energy produced for the two nuclides is mainly from alphaparticles and therefore only the alpha particles are considered in thefollowing estimate.

The ²¹²Pb and short-lived daughters produce on average 7.8 MeV alpharadiation per atom of ²¹²Pb. The half-life of ²¹²Pb is 10.6 h.

The ²¹³Bi and short-lived daughters produce on average 8.4 MeV of alphaenergy per atom of ²¹³Bi. The half-life of ²¹³Bi is 46 min.

It is assumed an equivalent dose for alpha particles of 5 Sv/Gy.

Thus 1 Bq of ²¹²Pb produce an equivalent alpha dose of1×(10.6×60/46)×7.8/8.4=12.6 Bq of ²¹³Bi when decaying completely.

It has been reported that salivary glands, kidneys and red marrow arethe dose limiting tissues for ²¹³Bi and ²²⁵Ac complexed to PSMA-617(Kratochwil, et al, 2018).

Based on the imaging of PSMA-617 labeled with radionuclides suitable forpositron emission tomography detection, it is assumed a maximumpercentage uptake to be at the 30 minutes time point, that 70% ²¹³Biatoms relative to 90% of ²¹²Pb would reach and decay in tumors (i.e.,the relative decay fraction).

The relative decay fraction for the dose limiting tissues is assumed tobe 70% for all the tissues with ²¹³Bi-PSMA-617. For ²¹²Pb-PSMA-617 takenup it is assumed that 50% would decay in the salivary glands, 30% inkidneys and 20% in bone marrow.

By correcting for energy per Bq and the relative decay fraction for²¹²Pb- and ²¹³Bi-labeled PSMA-617 the dose estimate for ²¹²Pb-PSMA-617would be as presented in Table 5 together with previously published datafor ²¹³Bi- and ²²⁵Ac-labeled PSMA-617. Also, by using mouse datacomparison of ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 and ²¹²Pb-PSMA-617 andassuming similar tissue uptake ratio in man dosimetry estimate for²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 is presented in Table 5.

TABLE 5 Dose assessment assuming similar stability and affinity ofproducts regardless of radionuclide. ²¹²Pb-p-SCN- ²²⁵Ac- ²¹³Bi- ²¹²Pb-Bn-TCMC- PSMA-617^(a) PSMA-617^(b) PSMA-617 PSMA ligand 1 Organ Sv/MBqSv/GBq Sv/100 MBq Sv/100 MBq^(c) Salivary 2.33 8.1 5.67 5.67 glandsKidneys 0.74 8.1 3.40 1.70 Red marrow 0.05 0.52 0.19 0.19 Tumors 5.7 6.310.2 10.2 ^(a)From Kratochwil et al 2017. ^(b)From Kratochwil et al2018. ^(c)Assuming same uptake as for ²¹²Pb-PSMA-617 exept for kidneyswhich is assumed to be reduced by 50%.

It has been reported that both ²²⁵Ac- and ²¹³Bi-labeled PSMA-617 hasbeen used clinically and shown considerable antitumor activity. Based onthe estimates in this example it is indicated that ²¹²Pb-labeled ureabased PSMA inhibitor is a very promising therapeutic tool against PSMAexpressing cancer.

Example 15 Dosimetry estimate for dual targeting ²²⁴Ra cation and²¹²Pb-labeled PSMA targeting urea derivative.

Using ²²⁴Ra in equilibrium with ²¹²Pb-labeled PSMA binding ureaderivative. For a dosage of 150 kBq of ²²⁴Ra the total activityadministered would be 10.5 MBq in a 70 kg person. By using publisheddosimetry for cationic ²²³Ra in prostate cancer patient and correctingfor half-life difference between ²²⁴Ra and ²²³Ra, assuming samebiodistribution, and considering the different residence times in thevarious tissues it was found that ²²⁴Ra would give per MBq administered0.006 Gy to kidneys, 0.029 Gy to salivary glands, 0.26 Gy to red marrowand anr estimated 5 times the red marrow uptake to tumors i.e, 1.3 Gy.

An equivalent dose of 5 Sv/Gy for alpha particle dose is assumed and thedata is translated to Sv per injected dosage (10.5 MBq/patient) in Table6.

TABLE 6 Dose assessment of ²²⁴Ra cation + ²¹²Pb-labeled PSMA bindingurea derivative*. ²²⁴Ra cation + ²¹²Pb-p-SCN-Bn- ²¹²Pb-p-SCN-Bn-TCMC-PSMA TCMC-PSMA ²²⁴Ra^(a) ligand ligand 1 Organ Sv/10.5 MBq 1Sv/10.5 MBq^(c) Sv/10.5 MBq Salivary glands 37 0.60 0.97 Kidneys 0.390.36 1.7  Red marrow^(b) 2.2 0.02 2.22 Tumors (bone 10.5/0.09 1.07/1.0711.6/1.16 metastases/soft tissue metastases) *Assuming 1:1 ratio between²²⁴Ra and ²¹²Pb. ^(a)From Lassmann et al, 2002. ^(b)It should be notedthat the red marrow is mainly irradiated from bone surface deposits ofRa, and due to short range of alpha-particles from the bone surfaces,substantial areas of red bone marrow are outside of reach from bonesurface alpha particles. Equivalent dose of 5 for alpha-particle dose isassumed.

Example 15. Dosimetry Estimated for ²²⁷Th-Labeled PSMA Binding UreaDerivative

In this example the ¹⁷⁷Lu is assumed labeled to p-SCN-Bn-DOTA-PSMAligand 2 or a HOPO derived version of this molecule. The currentestimates are based on adaptations of data from studies of ²²³Ra inpatients and ²²⁵Ac-PSMA-617 in patients with prostate cancer (Chittendenet al., 2015, Kratochwil et al, 2017, 2018).

An equivalent dose of 5 Sv per Gy is assumed for the alpha particleradiation.

It is assumed that the ²²³Ra generated in tissues would decay locally.It is assumed that 40% of the ²²³Ra generated from whole bodycirculation of ²²⁷Th is retained in the skeleton. Only the alphaparticle dose is considered as this constitute 95% or more of the totalradiation energy produced.

In tissue with a longer residence times for the radiolabeled PSMA ureaderivative (salivary gland, kidneys and tumors) the cumulated activityof ²²³Ra produced from ²²⁷Th is assumed to be 20% of the ²²⁷Th and forred bone marrow 5%.

The 20% and 5% radium generated from ²²⁷Th in the various tissues areassumed to be in equilibrium with alpha-emitting progeny radionuclides,so each radium decay in effect produces 26.4 MeV of alpha radiationwhile ²²⁷Th produces one alpha of 5.9 MeV.

Since residence times are lower than the half-lives of ²²⁵Ac and ²²⁷Th,the cumulated activity for ²²⁷Th is assumed to be only 10% higher thanfor ²²⁵Ac (not considering ²²⁵Ac progenies) in all organs. It is assumeda total of 27.7 MeV of alpha dose per atom of ²²⁵Ac from decay of themother nuclide and alpha emitting progenies.

It is also assumed that the red marrow dose and skeletal tumor dose isincreased by a factor of 2 vs. the red marrow dose, due to skeletaluptake of ²²³Ra generated during systemic circulation etc of the²²⁷Th-product.

The data are presented in Table 7. The data indicate favorable tumor totissue ratios for ²²⁷Th-labeled PSMA binding urea derivative.

TABLE 7 Dosimetry estimated of equivalent doses for critical organs andtumors for ²²⁷Th-labeled PSMA binding urea derivative (MBq/patient).²²³Ra (co-localized Organ ²²⁷Th (Sv/MBq) with ²²⁷Th) ²²⁷Th + ²²³RaSalivary glands 0.55 0.49 1.04 Kidneys 0.17 0.15 0.32 Red marrow  0.012 0.003  0.030* Tumors (soft 1.33/1.33 1.19/1.19 2.52/2.67* tissue/bonemet.) *Includes added skeletal dose of ²²³Ra generated from ²²⁷Th duringcirculation phase.

The data in Table 7 represent ²²⁷Th-labeled PSMA binding urea derivative(e.g. PSMA-617) which is purified from ²²³Ra prior to injection.Alternatively, it is possible to use the ²²⁷Th-product solution with apresence of various amounts of ²²³Ra to increase dose to the bonemetastases, e.g., if the bone disease is very dominating compared withsoft tissue disease.

It is assumed that the products described herein can be used in singletreatment or in repeated treatment fashion.

In conclusion, the dosimeric estimates for PSMA-targeting ureaderivatives labeled with ²¹²Pb or ²²⁷Th indicates promising tumor totissue ratios indicating that clinical benefit of use may be possible.

Example 16. Comparative Therapy Experiment with ¹⁷⁷Lu-PSMA-617 and²¹²Pb-p-SCN-Bn-TCMC-PSMA Ligand 1 in Nude Mice with C4-2 Xenografts

Male nude mice were inoculated in the flanks with C4-2 PSMA positivehuman prostate cells and 2 weeks later when tumors were 5-7 mm indiameters. Groups of 8 animals each received saline, 52 MBq of¹⁷⁷Lu-PSMA-617 or 320 kBq of ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1. Animalswere sacrificed when tumor size reached 20 mm due to animal welfarerequirements. Tumor dosimetry was calculated based on the followingassumptions: for ¹⁷⁷Lu-PSMA-617 effective half-life in tumor of 3 days,10% of injected dose per gram decays in tumor and 80% of radiation fromdecays in tumors is absorbed in tumors and 0.15 MeV radiation energy perdecay. For ²¹²Pb the effective half-life was assumed to be 10.6 h, 10%of injected dose per gram decays in tumor and 100% of radiation in atumor is absorbed in the tumor, 100% retention of ²¹²Pb and daughters inthe tumors and 8 MeV radiation energy per decay. The tumor dosimetry forthe injected activities gave on average 35.9 Gy to tumors for¹⁷⁷Lu-PSMA-617 and 2.06 Gy to tumors for ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand1 groups respectively.

Results: At day 30 after treatment the following the data showed 0%,12.5% and 75% survival in the saline, ¹⁷⁷Lu-PSMA-617 and²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 groups respectively (FIG. 2). Themedian survival was 15 days, 20 days and >30 days (not reached) in thesaline, ¹⁷⁷Lu-PSMA-617 and ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 groups,respectively.

In conclusion, the data indicates a strong tumor growth delay with²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 vs ¹⁷⁷Lu-PSMA-617 even though theradiation dose estimates show a 17 times higher radiation dose, in termsof Gy, delivered with ¹⁷⁷Lu-PSMA-617. Thus, the radiobiologicaleffectiveness (RBE) for ²¹²Pb-p-SCN-Bn-TCMC-PSMA ligand 1 vs¹⁷⁷Lu-PSMA-617 was at least 17. This high radiobiological effectivenessfor therapeutic levels of alpha emitter was highly unexpected as usuallyan RBE of 2-5 is expected for alpha emitters vs beta emitters.

REFERENCES

-   An efficient chelator for complexation of thorium-227.-   Ramdahl T, Bonge-Hansen H T, Ryan O B, Larsen S, Herstad G, Sandberg    M, Bjerke R M, Grant D, Brevik E M, Cuthbertson A S-   Bioorg Med Chem Lett. 2016 Sep. 1; 26(17):4318-21. In Vitro and In    Vivo Efficacy of a Novel CD33-Targeted Thorium-227 Conjugate for the    Treatment of Acute Myeloid Leukemia.-   Hagemann U B, Wickstroem K, Wang E, Shea A O, Sponheim K, Karlsson    J, Bjerke R M, Ryan O B, Cuthbertson A S-   Mol Cancer Ther. 2016 October; 15(10):2422-2431.-   Chittenden S J., A phase 1, open-label study of the biodistribution,    pharmacokinetics, and dosimetry of 223Ra-dicloride in patinets with    hormone-refractory prostate cancer and skeletal metastases. J Nucl    Med 56: 1304-1309 (2015).-   Kratochwil C et al., Targeted α-therapy of metastatic castration    resistant prostate cancer with 225Ac-PSMA-617: Dosimetry estimate    and empirical dose finding J Nucl Med 58: 1624-1631 (2017).-   Kratochwil C et al., Targeted a-therapy of mCRPC: Dosimetry estimate    of 213Bismuth-PSMA-617. Eur J Nucl Med Mol Imaging 45:31-37 (2018)-   Huang S S, Heston W D W. Should Low Molecular Weight PSMA Targeted    Ligands Get Bigger and Use Albumin Ligands for PSMA Targeting?    Theranostics 7: (7) 1940-1941 (2017)-   Choy C J et al., 177Lu-Labeled Phosphoramidate-Based PSMA    Inhibitors: The Effect of an Albumin Binder on Biodistribution and    Therapeutic Efficacy in Prostate Tumor-Bearing Mice. Theranostics.    2017; 7(7):1928-1939. doi:10.7150/thno.18719.-   Lassmann, M et al., Therapy of ankylosing spondylitis with    224Ra-radium chloride:Dosimetry and risk considerations. Radiat    Environ Biophys 41: 173-178 (2002).

Items

1. Compound X, wherein the compound X is a urea derivative suitable fortargeting of PSMA expressing cells and tissues.

2. A complex comprising a compound X linked to ²¹²Pb, ¹⁷⁷Lu, ²¹³Bi,²²⁵Ac or ²²⁷Th, wherein the compound X is a urea derivative suitable fortargeting of PSMA expressing cells and tissues.

3. The compound of item 1 or the complex according to item 2, whereinthe compound X is linked to ²¹²Pb or ²²⁷Th by a chelating moiety Z.

4. The compound or complex according to any of items 1-3, wherein thechelating moiety Z is selected from the group consisting of acyclicchelators, cyclic chelators, cryptands, crown ethers, porphyrins orcyclic or noncyclic polyphosphonates, DOTMP, EDTMP, bisphosphonate,DOTA, a DOTA derivative such as p-SCN-Bn-DOTA, pamidronate conjugated toDOTA, TCMC, a TCMC derivative such as p-SCN-Bn-TCMC, pamidronateconjugated to TCMC, antibody-conjugated-DOTA, antibody-conjugated-TCMC,HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA,DEDPA, and oxo-Do3A.

4. The compound or complex according to any of items 1-4, wherein thelinker is DOTA or a DOTA derivative.

5. The compound or complex according to any of items 1-4, wherein thelinker is a DOTA derivative such as p-SCN-Bn-DOTA.

6. The compound or complex according to any of items 1-4, wherein thelinker is TCMC or a TCMC derivative.

7. The compound or complex according to any of items 1-4 or 6, whereinthe linker is a TCMC derivative such as p-SCN-Bn-TCMC.

8. The compound or complex according to any of items 1-7, wherein thelinker an octadentate hydroxypyridinone-containing ligand, such as3,2-HOPO.

9. The compound or complex according to any of items 1-8, whereincompound X linked to a chelating moiety Z is defined by the formula I:

or a pharmaceutically acceptable salt thereof,

wherein

W is a PSMA-targeting ligand;

A⁴ is a bond or a divalent linking moiety comprising 1 to 10 carbonatoms in a chain, a ring, or a combination thereof, wherein at least onecarbon atom is optionally replaced with O, —NR³—, or —C(O)—;

G is C═O, C═S, C—NH₂, or C—NR³;

R¹ is hydrogen or a carboxylic acid protecting group;

R³ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,heterocycloalkyl, aryl, alkylaryl, and heteroaryl.

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are each independently hydrogen, alkyl,alkoxyl, or

R¹⁷ and R¹⁸ are each independently hydrogen, alkyl, aryl, or alkylaryl;

R¹⁹ is selected from the group consisting of alkyl, alkoxyl, halide,haloalkyl, and CN;

m is an integer from 1 to 6; and

o is an integer from 0 to 4, wherein when o is greater than 1, each R¹⁹is the same or different.

10. The compound or complex according to any of items 1 to 9, or apharmaceutically acceptable salt thereof, wherein

A⁴ is a bond, (CH₂)_(n), —HC(O)—, —(OCH₂CH₂)_(n)—, —(HCH₂CH₂)_(n)—,—H(CO)CH₂—, —HC(0)CH₂(OCH₂CH₂)_(n)—, or —HC(O)CH₂(HCH₂CH₂)_(n)—; and

L is a bond, (CH₂)_(n), —(OCH₂CH₂)_(n)—, —(HCH₂CH₂)_(n)—, or—C(O)(CH₂)_(n)—;

wherein n is independently 1, 2, or 3.

11. The compound or complex according to any of items 1-10, or apharmaceutically acceptable salt thereof, wherein A⁴ is a bond,—(OCH₂CH₂)_(n)—, or —HC(0)CH₂(OCH₂CH₂)_(n)—; and

L is a bond, or —(OCH₂CH₂)_(n)—;

wherein n is independently 1 or 2.

12. The compound or complex according to any of items 1-11, or apharmaceutically acceptable salt thereof, wherein

W has the structure:

wherein R²⁰ and R²¹ are each independently an amino acid residue linkedvia an amino group thereof to the adjacent —C(O)— group.

13. The compound or complex according to any of items 1-12, or apharmaceutically acceptable salt thereof, wherein

W has the structure:

wherein R is hydrogen or a carboxylic acid protecting group.

14. The compound or complex according to any of items 1-13, or apharmaceutically acceptable salt thereof, having the structure:

or a pharmaceutically acceptable salt thereof,

wherein R¹⁷ is aryl.

15. The compound or complex according to any of items 1-14, or apharmaceutically acceptable salt thereof, wherein the complex isPSMA-617:

With the radionuclide, such as ²¹²Pb, can be linked/chelated to the fourN.

16. The compound or complex according to item 15, wherein the DOTA unitis substituted with a TCMC unit.

17. The compound or complex according to items 15-16, wherein DOTA isp-SCN-Bn-DOTA and TCMC is p-SCN-Bn-TCMC.

18. The compound or complex according to items 15-17, whereinp-SCN-Bn-DOTA or p-SCN-Bn-TCMC are backbone C-linked to the ureaderivative (PSMA).

19. The compound or complex according to items 15-18, wherein thecompound is backbone-C linked p-SCN-Bn-DOTA or p-SCN-Bn-TCMC:

Wherein Z is:

And wherein X is: —OH or NH₂.

20. The compound or complex according to items 15-19, wherein thecompound is backbone-C linked p-SCN-Bn-DOTA or p-SCN-Bn-TCMC:

wherein X is: —OH or NH₂.

21. The compound or complex according to items 15-20, wherein thecompound is backbone-C linked p-SCN-Bn-DOTA i.e.p-SCN-Bn-DOTA-PSMA-ligand 2:

22. The compound or complex according to items 15-20, wherein thecompound is backbone-C linked p-SCN-Bn-TCMC i.e. p-SCN-Bn-TCMC-PSMAligand 1:

23. A PSMA targeting urea derivative containing a TCMC group forchelating ²¹²Pb.

24. A PSMA targeting urea derivative containing HOPO for chelating²²⁷Th.

25. A PSMA targeting urea derivative containing DOTA labeled with either²¹²Pb or ²²⁷Th.

26. A pharmaceutical composition comprising the compound or complexaccording to items 1-21, and/or a PSMA targeting urea derivativeaccording to claims 15-17, and a diluent, carrier, surfactant, and/orexcipient.

27. The radiopharmaceutical composition according to item 26, furthercomprising ²²⁴Ra.

28. The radiopharmaceutical composition according to any of the items26-27, wherein the radioactivity is 100 kBq to 100 MBq per done.

29. The radiopharmaceutical composition according to any of items 26-28,wherein the amount of ²²⁴Ra and ²¹²Pb is in radioactive equilibrium.

30. The radiopharmaceutical composition according to any of items 26-29,wherein the activity ratio (MBq) between ²¹²Pb to ²²⁴Ra is between 0.5and 2, such as 0.8-1.5, or such as 0.8-1.3, or preferably such as0.9-1.15.

31. A kit comprising:

-   -   a first vial comprising a radiopharmaceutical composition        according to any of items 26-30, and    -   a second vial comprising a neutralizing solution to adjust pH        and/or isotonicity of the radiopharmaceutical composition prior        to administration to a patient.

32. A kit comprising:

-   -   a first vial comprising a solution comprising ²²⁴Ra, ²¹²Pb        and/or ²²⁷Th;    -   a second vial comprising a complexing agent selected from the        group consisting of acyclic chelators, cyclic chelators,        cryptands, crown ethers, porphyrins or cyclic or noncyclic        polyphosphonates, DOTMP, EDTMP, bisphosphonate derivatives,        DOTA, a DOTA derivative, pamidronate conjugated to DOTA, TCMC, a        TCMC derivative, pamidronate conjugated to TCMC,        antibody-conjugated-DOTA, antibody-conjugated-TCMC, HBED-CC,        NOTA, NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA,        DEDPA, and oxo-Do3A, or compound according to any of items 1-21,        wherein the complexing agent is capable of complexing a daughter        nuclide of ²²⁴Ra, such as ²¹²Pb, and wherein the complexing        agent does not complex ²²⁴Ra in the pharmaceutical solution; and    -   optionally, instructions for mixing the first vial and the        second vial, thereby forming a pharmaceutical composition ready        to be administered to a patient 1 minute to 12 hours after        mixing.

33. The kit according to any of claims 31-32, wherein the kit is for useas a medicament.

34. The radiopharmaceutical composition according to any of items 18-22for use as a medicament.

35. The radiopharmaceutical composition according to any of items 18-22for use in the treatment of skeletal disease.

36. The radiopharmaceutical composition for use according to item 35,wherein the skeletal disease is selected from the group consisting ofskeletal metastases from cancers to the breast, prostate, kidneys, lung,bone, or multiple myeloma, or non-cancerous diseases causing undesiredcalcification including ankylosing spondylitis.

37. The radiopharmaceutical composition for use according to any ofitems 34-36, wherein the solution is administered at a dose in the range50-150 kBq per kg of bodyweight, such as 50-100 kBq per kg ofbodyweight.

38. A method of treatment of malignant or non-malignant disease byadministration of a radiopharmaceutical composition as described initems 26-30 to an individual in need thereof.

39. A method for providing a radiopharmaceutical composition accordingto any of items 26-30, the method comprising:

a) providing a first solution wherein the amount of ²²⁴Ra and ²¹²Pb isin radioactive equilibrium;

b) providing a second solution comprising a complexing agent that isselected from the group consisting of acyclic chelators, cyclicchelators, cryptands, crown ethers, porphyrins or cyclic or noncyclicpolyphosphonates, DOTMP, EDTMP, bisphosphonate, DOTA, a DOTA derivative,pamidronate conjugated to DOTA, TCMC, a TCMC derivative, pamidronateconjugated to TCMC, antibody-conjugated-DOTA, antibody-conjugated-TCMC,HBED-CC, NOTA, NODAGA, TRAP, NOPO, PCTA, DFO, DTPA, CHX-DTPA, AAZTA,DEDPA, and oxo-Do3A, wherein the complexing agent is capable ofcomplexing a daughter nuclide of ²²⁴Ra; such as ²¹²Pb, and wherein thecomplexing agent does not complex ²²⁴Ra; and

c) mixing the first composition and the second composition, therebyproviding a pharmaceutical composition according to any of item 26-30.

1. A compound comprising a PSMA unit and chelating unit, wherein thePSMA unit comprises a carbon-backbone linked to the chelating unit, andwherein the compound is of the formula:

wherein X is: —NH₂ or —OH. 2-18. (canceled)
 19. The compound accordingto claim 1, wherein the compound is:


20. The compound according to claim 1, wherein the compound is:


21. A complex comprising the compound according to a claim 1 complexedwith a radionuclide selected from the group consisting of ²¹²Pb, ²¹²Bi,²¹³Bi, ²²⁵Ac and ²²⁷Th.
 22. The compound according to claim 19, whereinsaid compound is complexed with ²¹²Pb.
 23. The compound according toclaim 20, wherein said compound is complexed with ²¹²Pb, ²¹²Bi, ²¹³Bi,²²⁵Ac or ²²⁷Th.
 24. A radiopharmaceutical composition comprising thecompound according to claim 1, a radionuclide, and a diluent, carrier,surfactant, and/or excipient.
 25. The radiopharmaceutical compositionaccording to claim 24, further comprising ²²⁴Ra.
 26. Theradiopharmaceutical composition according to claim 25, furthercomprising ²¹²Pb and, wherein the amount of ²²⁴Ra and ²¹²Pb is inradioactive equilibrium.
 27. The radiopharmaceutical compositionaccording to claim 26, wherein the activity ratio (MBq) between ²¹²Pb to²²⁴Ra is between 0.5 and
 2. 28. The radiopharmaceutical compositionaccording to claim 24, wherein composition is dosaged with aradioactivity of 100 kBq to 100 MBq per dose.
 29. A method of inhibitingor treating a disease in a subject comprising administering theradiopharmaceutical of claim 24 to a subject in need thereof.
 30. Themethod of claim 29, wherein said disease is a malignancy of the softtissue or skeletal system.
 31. The method according to claim 30, whereinthe malignancy of the skeletal system is selected from skeletalmetastases from cancers to the breast, prostate, kidneys, lung, bone, ormultiple myeloma.
 32. The method according to claim 31, wherein theradiopharmaceutical is administered at a dose in the range 50-150 kBqper kg of bodyweight.
 33. A kit comprising: a first vial comprising aradiopharmaceutical composition according to claim 24, and a second vialcomprising a neutralizing solution to adjust pH and/or isotonicity ofthe radiopharmaceutical composition prior to administration to apatient.
 34. A kit comprising: a first vial comprising a solutioncomprising ²²⁴Ra, ²¹²Pb and/or ²²⁷Th; a second vial comprising acompound according to claim 1.